There is an urgent clinical need for the development of opioid analgesics with novel biological activity profiles that lack the limiting side effects of currently available opiates. This project concerns the development of ? opioid agonists and of compounds with mixed ?/? activity profiles as pharmacological tools and as analgesics expected to produce fewer side effects. In Specific Aim 1, ? opioid peptide agonists containing novel phenylalanine analogues in place of tyrosine-1 will be synthesized. These compounds have been designed to have distinct ? opioid receptor binding modes and to induce or recognize a distinct receptor conformation. Therefore, these compounds may behave as functionally selective or biased ? agonists with regard to receptor signaling or internalization and have potential as analgesics with reduced side effects (e.g. tolerance development). Some of these peptides will be prepared in glycosylated form to improve blood-brain barrier (BBB) permeability. The compounds will be pharmacologically characterized in opioid receptor binding assays and functional assays, ? receptor phosphorylation and internalization studies, electrophysiological studies (Ca2+ channel activity) and bioluminescence resonance energy transfer (BRET) experiments for the identification of ? receptor interactions with various signaling effectors and -arrestin. Two inflammatory pain models will be used to determine antinociceptive potencies and analgesic tolerance development. Specific Aim 2 is based on the observation that co-administered ? and ? opioid agonists act synergistically to produce a potent antinociceptive effect, thus providing the rationale for the development of mixed ? agonist/? agonists as centrally acting analgesics producing fewer side effects. It is proposed to prepare novel bifunctional compounds incorporating various ? opioid agonists linked to the opioid peptide [Dmt1]DALDA which has a dual role as ? agonist component and as BBB-penetrating vector. Compounds will be characterized in vitro and their antinociceptive potencies will be determined in acute pain models. Their propensities to produce analgesic tolerance, physical dependence, constipation and respiratory depression will be examined. Specific Aim 3 concerns the continued development of mixed ? agonist/? antagonists with demonstrated potential as centrally acting analgesics having low propensity to produce the typical side effects of ? agonists like morphine. One design of such bifunctional compounds makes again use of [Dmt1]DALDA as ? agonist component and BBB-penetrating vector to which various ? neutral antagonists, inverse agonists or partial agonists will be linked. In another design glycosylated analogues of the ? agonist/? antagonist DIPP-NH2[?] will be prepared in an effort to improve its ability to cross the BBB. The in vitro and in vivo pharmacological characterization will be as in Aim 2.